Autographa Californica Multiple Nucleopolyhedrovirus P48 (Ac103) Is Required for the Efficient Formation of Virus-Induced Intranuclear Microvesicles

Our previous study has shown that the Autographa californica multiple nucleopolyhedrovirus(AcMNPV) p48(ac103)gene is essential for the nuclear egress of nucleocapsids and the formation of occlusion-derived virions(ODVs). However,the exact role of p48 in the morphogenesis of ODVs remains unknown. In this study, we demonstrated that p48 was required for the efficient formation of intranuclear microvesicles. To further understand its functional role in intranuclear microvesicle formation, we characterized the distribution of the P48 protein, which was found to be associated with the nucleocapsid and envelope fractions of both budded virions and ODVs. In Ac MNPV-infected cells, P48 was predominantly localized to nucleocapsids in the virogenic stroma and the nucleocapsids enveloped in ODVs, with a limited but discernible distribution in the plasma membrane, nuclear envelope, intranuclear microvesicles, and ODV envelope. Furthermore,coimmunoprecipitation assays showed that among the viral proteins required for intranuclear microvesicle formation, P48 associated with Ac93 in the absence of viral infection.     

Identification of Autographa californica nucleopolyhedrovirus ac93 as a core gene and its requirement for intranuclear microvesicle formation and nuclear egress of nucleocapsids

Autographa californica nucleopolyhedrovirus (AcMNPV) orf93 (ac93) is a highly conserved uncharacterized gene that is found in all of the sequenced baculovirus genomes except for Culex nigripalpus NPV. In this report, using bioinformatics analyses, ac93 and odv-e25 (ac94) were identified as baculovirus core genes and thus p33-ac93-odv-e25 represent a cluster of core genes. To investigate the role of ac93 in the baculovirus life cycle, an ac93 knockout AcMNPV bacmid was constructed via homologous recombination in Escherichia coli. Fluorescence and light microscopy showed that the AcMNPV ac93 knockout did not spread by infection, and titration assays confirmed a defect in budded virus (BV) production. However, deletion of ac93 did not affect viral DNA replication. Electron microscopy indicated that ac93 was required for the egress of nucleocapsids from the nucleus and the formation of intranuclear microvesicles, which are precursor structures of occlusion-derived virus (ODV) envelopes. Immunofluorescence analyses showed that Ac93 was concentrated toward the cytoplasmic membrane in the cytoplasm and in the nuclear ring zone in the nucleus. Western blot analyses showed that Ac93 was associated with both nucleocapsid and envelope fractions of BV, but only the nucleocapsid fraction of ODV. Our results suggest that ac93, although not previously recognized as a core gene, is one that plays an essential role in the formation of the ODV envelope and the egress of nucleocapsids from the nucleus.     

Baculovirus Data Suggest a Common but Multifaceted Pathway for Sorting Proteins to the Inner Nuclear Membrane

Multiple unique protein markers sorted to the inner nuclear membrane (INM) from the Autographa californica nucleopolyhedrovirus occlusion-derived virus (ODV) envelope were used to decipher common elements of the sorting pathway of integral membrane proteins from their site of insertion into the membrane of the endoplasmic reticulum (ER) through their transit to the INM. The data show that during viral infection, the viral protein FP25K is a partner for all known ODV envelope proteins and that BV/ODV-E26 (designated E26) is a partner for some, but not all, such proteins. The association with the ER membrane of FP25K, E26, and the cellular INM-sorting protein importin-α-16 is not static; rather, these sorting proteins are actively recruited to the ER membrane based upon requirements of the proteins in transit to the INM. Colocalization analysis using an ODV envelope protein and importin-α-16 shows that during viral infection, importin-α-16 translocates across the pore membrane to the INM and then is incorporated into the virus-induced intranuclear membranes. Thus, the association of importin-α-16 and INM-directed proteins appears to remain at least through protein translocation across the pore membrane to the INM. Overall, the data suggest that multiple levels of regulation facilitate INM-directed protein trafficking, and that proteins participating in this sorting pathway have a dynamic relationship with each other and the membrane of the ER.     

Occlusion-derived baculovirus: interaction with human cells and evaluation of the envelope protein P74 as a surface display platform

To develop complementary baculovirus-based tools for gene delivery and display technologies, the interaction of occlusion-derived baculovirus (ODV) with human cells, and the functionality of the P74 ODV envelope protein for display of the IgG-binding Z domains (ZZP74) were evaluated. The cellular binding of ODV was concentration-dependent and saturable. Only minority of the bound virions were internalized at both 37 and 4 degrees C, suggesting usage of direct membrane fusion as the entry mode. The intracellular transport of ODV was confined in vesicular structures peripheral to the plasma membrane, impeding subsequent nuclear entry and transgene expression. Transduction of ODV was not rescued by mimicking the preferred alkaline environment and lowered temperature of the ODV infective entry, or following treatment with the microtubule depolymerizing agent nocodazole or with the histone deacetylase inhibitor sodium butyrate. Similar to unmodified P74, the ZZP74 chimera localized in the intranuclear ring zone, and was enriched in virus-induced microvesicles. However, Western blotting of ODV and budded virions (BV), as well as viral envelope and nucleocapsid fractions combined with functional infection/transduction studies revealed incorporation of the ZZP74 fusion protein into viral nucleocapsids. The ZZP74 BV preserved normal infectivity, polypeptide profile, and morphology, but became incapable of entering and transducing human cells.     

The Baculovirus Core Gene ac83 Is Required for Nucleocapsid Assembly and Per Os Infectivity of Autographa californica Nucleopolyhedrovirus

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac83 is a baculovirus core gene whose function in the AcMNPV life cycle is unknown. In the present study, an ac83-knockout AcMNPV (vAc83KO) was constructed to investigate the function of ac83 through homologous recombination in Escherichia coli. No budded virions were produced in vAc83KO-transfected Sf9 cells, although viral DNA replication was unaffected. Electron microscopy revealed that nucleocapsid assembly was aborted due to the ac83 deletion. Domain-mapping studies revealed that the expression of Ac83 amino acid residues 451 to 600 partially rescued the ability of AcMNPV to produce infectious budded virions. Bioassays indicated that deletion of the chitin-binding domain of Ac83 resulted in the failure of oral infection of Trichoplusia ni larvae by AcMNPV, but AcMNPV remained infectious following intrahemocoelic injection, suggesting that the domain is involved in the binding of occlusion-derived virions to the peritrophic membrane and/or to other chitin-containing insect tissues. It has been demonstrated that Ac83 is the only component with a chitin-binding domain in the per os infectivity factor complex on the occlusion-derived virion envelope. Interestingly, a functional inner nuclear membrane sorting motif, which may facilitate the localization of Ac83 to the envelopes of occlusion-derived virions, was identified by immunofluorescence analysis. Taken together, these results demonstrate that Ac83 plays an important role in nucleocapsid assembly and the establishment of oral infection.     

The ha72 Core Gene of Baculovirus Is Essential for Budded Virus Production and Occlusion-Derived Virus Embedding,and Amino Acid K22 Plays an Important Role in Its Function

ha72 of Helicoverpa armigera nucleopolyhedrovirus (a homologue of ac78) was identified as a conserved late baculovirus gene and characterized. HA72 localizes in the intranuclear ring zone. By generating mutants, we showed that HA72 is essential for budded virus (BD) production and occlusion-derived virus (ODV) embedding. HA72 also interacted with P33, a baculoviral sulfhydryl oxidase. A point mutation of amino acid 22 from lysine to glutamic acid curtailed BV production and precluded ODV occlusion as well as interaction with P33.     

Morphogenesis of Pestiviruses: New Insights from Ultrastructural Studies of Strain Giraffe-1

Knowledge on the morphogenesis of pestiviruses is limited due to low virus production in infected cells. In order to localize virion morphogenesis and replication sites of pestiviruses and to examine intracellular virion transport, a cell culture model was established to facilitate ultrastructural studies. Based on results of virus growth kinetic analysis and quantification of viral RNA, pestivirus strain Giraffe-1 turned out to be a suitable candidate for studies on virion generation and export from culture cells. Using conventional transmission electron microscopy and single-tilt electron tomography, we found virions located predominately in the lumen of the endoplasmic reticulum (ER) in infected cells and were able to depict the budding process of virions at ER membranes. Colocalization of the viral core protein and the envelope glycoprotein E2 with the ER marker protein disulfide isomerase (PDI) was demonstrated by immunogold labeling of cryosections. Moreover, pestivirions could be shown in transport vesicles and the Golgi complex and during exocytosis. Interestingly, viral capsid protein and double-stranded RNA (dsRNA) were detected in multivesicular bodies (MVBs), which implies that the endosomal compartment plays a role in pestiviral replication. Significant cellular membrane alterations such as those described for members of the Flavivirus and Hepacivirus genera were not found. Based on the gained morphological data, we present a consistent model of pestivirus morphogenesis.     

The Autographa californica Multiple Nucleopolyhedrovirus ORF78 Is Essential for Budded Virus Production and General Occlusion Body Formation

ORF78 (ac78) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a baculovirus core gene of unknown function. To determine the role of ac78 in the baculovirus life cycle, an AcMNPV mutant with ac78 deleted, Ac78KO, was constructed. Quantitative PCR analysis revealed that ac78 is a late gene in the viral life cycle. After transfection into Spodoptera frugiperda cells, Ac78KO produced a single-cell infection phenotype, indicating that no infectious budded viruses (BVs) were produced. The defect in BV production was also confirmed by both viral titration and Western blotting. However, viral DNA replication was unaffected, and occlusion bodies were formed. An analysis of BVs and occlusion-derived viruses (ODVs) revealed that AC78 is associated with both forms of the virions and is an envelope structural protein. Electron microscopy revealed that AC78 also plays an important role in the embedding of ODV into the occlusion body. The results of this study demonstrate that AC78 is a late virion-associated protein and is essential for the viral life cycle.     

Characterization of the <Emphasis Type="Italic">Bm61</Emphasis> of the <Emphasis Type="Italic">Bombyx mori</Emphasis> Nucleopolyhedrovirus

orf61 (bm61) of Bombyx mori Nucleopolyhedrovirus (BmNPV) is a highly conserved baculovirus gene, suggesting that it performs an important role in the virus life cycle whose function is unknown. In this study, we describe the characterization of bm61. Quantitative polymerase chain reaction (qPCR) and western blot analysis demonstrated that bm61 was expressed as a late gene. Immunofluorescence analysis by confocal microscopy showed that BM61 protein was localized on nuclear membrane and in intranuclear ring zone of infected cells. Structure localization of the BM61 in BV and ODV by western analysis demonstrated that BM61 was the protein of both BV and ODV. In addition, our data indicated that BM61 was a late structure protein localized in nucleus.     

Expression of DNAJB12 or DNAJB14 Causes Coordinate Invasion of the Nucleus by Membranes Associated with a Novel Nuclear Pore Structure

DNAJB12 and DNAJB14 are transmembrane proteins in the endoplasmic reticulum (ER) that serve as co-chaperones for Hsc70/Hsp70 heat shock proteins. We demonstrate that over-expression of DNAJB12 or DNAJB14 causes the formation of elaborate membranous structures within cell nuclei, which we designate DJANGOS for DNAJ-associated nuclear globular structures. DJANGOS contain DNAJB12, DNAJB14, Hsc70 and markers of the ER lumen and ER and nuclear membranes. Strikingly, they are evenly distributed underneath the nuclear envelope and are of uniform size in any one nucleus. DJANGOS are composed primarily of single-walled membrane tubes and sheets that connect to the nuclear envelope via a unique configuration of membranes, in which the nuclear pore complex appears anchored exclusively to the outer nuclear membrane, allowing both the inner and outer nuclear membranes to flow past the circumference of the nuclear pore complex into the nucleus. DJANGOS break down rapidly during cell division and reform synchronously in the daughter cell nuclei, demonstrating that they are dynamic structures that undergo coordinate formation and dissolution. Genetic studies showed that the chaperone activity of DNAJ/Hsc70 is required for the formation of DJANGOS. Further analysis of these structures will provide insight into nuclear pore formation and function, activities of molecular chaperones, and mechanisms that maintain membrane identity.     

CRUCIFORM NUCLEAR DIVISION IN SOROSPHAERA VERONICAE

Sorosphaera veronicae Schroet. is an endobiotic, holocarpic, obligately parasitic fungus presently classified in the Plasmodiophoromycetes. The ultrastructure of nuclear envelope formation in somatic nuclear division in cystosoral plasmodia was studied. The inner membrane of the nuclear envelope during prophase appears to invaginate and blebb off intranuclear membranous vesicles. The intranuclear membranous vesicles become associated with the surface of the separating chromatin in anaphase and eventually are involved in the formation of daughter nuclear envelopes within the original nuclear envelope. The sequence of nuclear envelope breakdown and reformation in S. veronicae is noteworthy because it emphasizes alternate methods of nuclear envelope formation other than the generally considered “typical” formation described in Allium cepa L.     

The amino-terminal domain of the lamin B receptor is a nuclear envelope targeting signal

The lamin B receptor (LBR) is a polytopic protein of the inner nuclear membrane. It is synthesized without a cleavable amino-terminal signal sequence and composed of a nucleoplasmic amino-terminal domain of 204 amino acids followed by a hydrophobic domain with eight putative transmembrane segments. To identify a nuclear envelope targeting signal, we have examined the cellular localization by immunofluorescence microscopy of chicken LBR, its amino-terminal domain and chimeric proteins transiently expressed in transfected COS-7. Full- length LBR was targeted to the nuclear envelope. The amino-terminal domain, without any transmembrane segments, was transported to the nucleus but excluded from the nucleolus. When the amino-terminal domain of LBR was fused to the amino-terminal side of a transmembrane segment of a type II integral membrane protein of the ER/plasma membrane, the chimeric protein was targeted to the nuclear envelope, likely the inner nuclear membrane. When the amino-terminal domain was deleted from LBR and replaced by alpha-globin, the chimeric protein was retained in the ER. These findings demonstrate that the amino-terminal domain of LBR is targeted to the nucleus after synthesis in the cytoplasm and that this polypeptide can function as a nuclear envelope targeting signal when located at the amino terminus of a type II integral membrane protein synthesized on the ER.     

Autographa californica Multiple Nucleopolyhedrovirus Core Gene ac96 Encodes a Per Os Infectivity Factor (pif-4)

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac96 is a core gene, but its role in virus replication is still unknown. To determine its role in the baculovirus life cycle, we used the AcMNPV bacmid system to generate an ac96-null virus (vAc^96null). Our analyses showed that the absence of ac96 does not affect budded virus (BV) production or viral DNA replication in infected Sf9 cells. Western blotting and confocal immunofluorescence analysis showed that AC96 is expressed in both the cytoplasm and the nucleus throughout infection. In addition, AC96 was detected in the envelope fractions of both BV and occlusion-derived virus. Injection of vAc^96null BV into the hemocoel killed Trichoplusia ni larvae as efficiently as repaired and control viruses; however, vAc^96null was unable to infect the midgut tissue of Trichoplusia ni larvae when inoculated per os. Therefore, the results of this study show that ac96 encodes a new per os infectivity factor (PIF-4).The Baculoviridae comprise a large and diverse group of viruses that are pathogens of insects, mainly from the Lepidoptera, Hymenoptera, and Diptera. During the typical biphasic infection cycle, two structurally and functionally distinct enveloped virion phenotypes are produced: occlusion-derived virus (ODV) and budded virus (BV) (35). The primary infection cycle in animals begins in the midgut cell after occlusion bodies (OBs) are ingested. Upon ingestion, the OBs dissolve in the alkaline environment of the midgut, and the ODVs are released into the lumen of midgut (15, 16, 20). Virions pass through a disrupted peritrophic membrane, a process often facilitated by enhancins, a group of virus-encoded metalloproteases (38). Subsequently, ODVs bind to and fuse directly with the microvilli of midgut columnar epithelial cells. A protein receptor is proposed to mediate the process, since binding is proteinase sensitive and saturable (15, 16, 20). After the nucleocapsids are transported to the nuclei of the midgut cells, viral DNA is released, followed by gene expression, DNA replication, and assembly of progeny nucleocapsids. In the late phase of infection, newly formed nucleocapsids are transported to the cell membrane, bud from the cell, and acquire a new envelope from the basal membrane. The BVs spread via the hemolymph (16) and the tracheal system (8) into the other tissues of the insect, causing the secondary infection.Baculoviruses encode per os infectivity factors (PIFs) on the envelope surface of ODV to initiate the efficient primary infection in midgut. So far, four highly conserved core genes, p74 (pif-0), pif-1, pif-2, and pif-3, have been identified. The deletion of the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) p74 gene results in the complete elimination of the per os infectivity of OBs, while virions purified from mutant OBs were infectious when injected into the hemocoels of Trichoplusia ni or Heliothis virescens larvae (13, 17, 22). P74 is proposed to function as an ODV attachment protein that binds to a specific 30-kDa receptor protein on the primary target cells within the midgut (17, 39). PIF-1 was originally identified in Spodoptera littoralis NPV, where the deletion of pif-1 (spli7) resulted in viruses that were unable to infect S. littoralis larvae per os (21). PIF-2 was first identified in Spodoptera exigua MNPV, and the disruption of pif-2 resulted in the complete loss of per os infectivity for the host (11, 31). PIF-1 and PIF-2 have also been shown to participate in the binding of ODV to target cells in the midgut (28). PIF-3 (ac115) is also an essential factor for oral infection of AcMNPV. Although PIF-3 is not required for ODV attachment and fusion, it may mediate a critical downstream event, such as the translocation of ODV along microvilli during primary infection (28).AcMNPV, the archetype Alphabaculovirus of the Baculoviridae, has a double-stranded DNA genome of approximately 134 kbp that contains 154 predicted open reading frames (ORFs) (1). Comparative analysis of the 49 completely sequenced baculovirus genomes reveals 31 core genes that are conserved in all baculovirus genomes and are therefore likely to serve important roles in baculovirus life cycles (14, 26, 32, 37). Most core genes are related either to DNA replication, gene expression, packaging and assembly, or per os infection (37). Four core genes, ac68, p33 (ac92), ac96, and ac109, still have no known function or sequence similarities to proteins of known functions.In this study, an ac96-null mutant was constructed utilizing an AcMNPV bacmid, and the results showed that in tissue culture, ac96 was nonessential and was not required for viral DNA replication, ODV production, or BV production. However, in vivo assays demonstrated that the ac96-null virus was unable to infect midgut tissue when T. ni larvae were inoculated per os. The core gene ac96 therefore encodes a new per os infectivity factor, PIF-4.     

Integral Membrane Proteins of the Nuclear Envelope Are Dispersed throughout the Endoplasmic Reticulum during Mitosis

We have analyzed the fate of several integral membrane proteins of the nuclear envelope during mitosis in cultured mammalian cells to determine whether nuclear membrane proteins are present in a vesicle population distinct from bulk ER membranes after mitotic nuclear envelope disassembly or are dispersed throughout the ER. Using immunofluorescence staining and confocal microscopy, we compared the localization of two inner nuclear membrane proteins (laminaassociated polypeptides 1 and 2 [LAP1 and LAP2]) and a nuclear pore membrane protein (gp210) to the distribution of bulk ER membranes, which was determined with lipid dyes (DiOC₆ and R6) and polyclonal antibodies. We found that at the resolution of this technique, the three nuclear envelope markers become completely dispersed throughout ER membranes during mitosis. In agreement with these results, we detected LAP1 in most membranes containing ER markers by immunogold electron microscopy of metaphase cells. Together, these findings indicate that nuclear membranes lose their identity as a subcompartment of the ER during mitosis. We found that nuclear lamins begin to reassemble around chromosomes at the end of mitosis at the same time as LAP1 and LAP2 and propose that reassembly of the nuclear envelope at the end of mitosis involves sorting of integral membrane proteins to chromosome surfaces by binding interactions with lamins and chromatin.     

A link between the synthesis of nucleoporins and the biogenesis of the nuclear envelope

The nuclear pore complex (NPC) is a multicomponent structure containing a subset of proteins that bind nuclear transport factors or karyopherins and mediate their movement across the nuclear envelope. By altering the expression of a single nucleoporin gene, NUP53, we showed that the overproduction of Nup53p altered nuclear transport and had a profound effect on the structure of the nuclear membrane. Strikingly, conventional and immunoelectron microscopy analysis revealed that excess Nup53p entered the nucleus and associated with the nuclear membrane. Here, Nup53p induced the formation of intranuclear, tubular membranes that later formed flattened, double membrane lamellae structurally similar to the nuclear envelope. Like the nuclear envelope, the intranuclear double membrane lamellae enclosed a defined cisterna that was interrupted by pores but, unlike the nuclear envelope pores, they lacked NPCs. Consistent with this observation, we detected only two NPC proteins, the pore membrane proteins Pom152p and Ndc1p, in association with these membrane structures. Thus, these pores likely represent an intermediate in NPC assembly. We also demonstrated that the targeting of excess Nup53p to the NPC and its specific association with intranuclear membranes were dependent on the karyopherin Kap121p and the nucleoporin Nup170p. At the nuclear envelope, the abilities of Nup53p to associate with the membrane and drive membrane proliferation were dependent on a COOH-terminal segment containing a potential amphipathic alpha-helix. The implications of these results with regards to the biogenesis of the nuclear envelope are discussed.     

Frog Virus 3 Open Reading Frame 97R Localizes to the Endoplasmic Reticulum and Induces Nuclear Invaginations

Frog virus 3 (FV3) is the type species of the genus Ranavirus, family Iridoviridae. The genome of FV3 is 105,903 bases in length and encodes 97 open reading frames (ORFs). The FV3 ORF 97R contains a B-cell lymphoma 2 (Bcl-2) homology 1 (BH1) domain and has sequence similarity to the myeloid cell leukemia-1 (Mcl-1) protein, suggesting a potential role in apoptosis. To begin to understand the role of 97R, we characterized 97R through immunofluorescence and mutagenesis. Here we demonstrated that 97R localized to the endoplasmic reticulum (ER) at 24 h posttransfection. However, at 35 h posttransfection, 97R localized to the ER but also began to form concentrated pockets continuous with the nuclear membrane. After 48 h posttransfection, 97R was still localized to the ER, but we began to observe the ER and the outer nuclear membrane invaginating into the nucleus. To further explore 97R targeting to the ER, we created a series of C-terminal transmembrane domain deletion mutants. We found that deletion of 29 amino acids from the C terminus of 97R abolished localization to the ER. In contrast, deletion of 12 amino acids from the C terminus of 97R did not affect 97R localization to the ER. In addition, a hybrid protein containing the 97R C-terminal 33 amino acids was similarly targeted to the ER. These data indicate that the C-terminal 33 amino acids of 97R are necessary and sufficient for ER targeting.